4 replies to this topic

[greenhorn]

Hello everyone,

I am trying to establish a cell line stably over-expressing my gene of interest. Transfecting the cDNA in a pcDNA6-plasmid lead to apoptosis of the cells with a 3 day delay compared to untransfected controls. Is there any good way to demonstrate clearly that this is sequence specific and definitely not related to transfection efficiency? Or a first step before I'd start cloning my cDNA into other (i.e. inducible) systems or similiar?

Any help would be really appreciated!!

[bob1]

Did you do an "empty" vector control (i.e. pcDNA6 with no insert) and a non-target vector control (i.e. pcDNA6 with another cDNA or a scrambled version of your cDNA)?

[greenhorn]

bob1, on 22 June 2011 - 06:08 PM, said:

Did you do an "empty" vector control (i.e. pcDNA6 with no insert) and a non-target vector control (i.e. pcDNA6 with another cDNA or a scrambled version of your cDNA)?

Hello,

Yes, I had the empty vector, which was proliferating happily under selection but no scrambled cDNA. How could I make a scrambled version of my cDNA?

Thanks so much for any help!

[bob1]

You can use another gene as a "non-target" control - if the cells die when you transfect this in, then it is a transfection issue, not a property of your gene.

You should also check expression of your gene in the cells by western and/or RT-PCR.

[greenhorn]

Thank you very much! I will measure mRNA expression and transfect another gene in parallel as a first step ).